Proper function of the synapse relies on a remarkably precise assemblage of molecular and morphological specializations. However, while much has been learned about the biochemical, structural and physiological features which characterize the mature synapse, much less is known about the mechanisms which direct synapse formation. Agrin is an extracellular matrix protein derived from Torpedo electric organ that organizes acetylcholine receptor (AchR) and acetylcholinesterase (AchE) on cultured myotubes. At the mature neuromuscular junction, immunocytochemical studies have shown that molecules closely related to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a central part in directing synapse regeneration. These results strongly suggest that agrin- related molecules play a role in directing the formation of the regenerating nerve-muscle synapse. The goal of the studies proposed here is to determine how agrin- related molecules might be involved in the differentation of the developing synapse. The experimental plan is to use recently developed monoclonal antibodies to Torpedo agrin to establish the time of appearance and the distribution of agrin-related molecules at the developing chick neuromuscular synapse in vivo. These antibodies will be used in concert with other markers to correlate the localization and expression of agrin-related molecules with other key events in synapse formation, such as the onset of functional innervation and the formation of high density clusters of AchR and AchE. The relationship of the expression and localization of agrin-related molecules to innervation will be investigated in experiments utilizing paralyzed and aneural muscles. These studies should yield important insights into the molecular mechanisms which underlie synapse formation in the developing embryo, and could provide important clues toward developing effective treatments for diseases and trauma which affect the neuromuscular synapse.